Detection of P. larvae in Local Honey

From 3-8-15.
Also from Tripartite conjugation from 1-7-15.

Ran PCR of several different isolates using the AFB primer sets. Crude genomic DNA was used as template DNA for the reaction. I was running low on PCR super mix and really had to skimp on volumes which may have effected accuracy.

Top:
Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Hi Lo	8a	2a	22a	63a	Q	R. Trach	None
Bottom:
Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
	Hi Lo	TransConj	TransConj	TransConj	9545	Neg

Discussion

There was apparently an issue when loading/running the gel as evident by the smears near the wells. The obvious problem appears to be that there is way too much DNA in the wells. My positive control, P. larvae 9545 in Lane 7 of the bottom row, checked out no problem. It is about the correct size as it should be and has a clear and definite band. However, most of the other bands are not so clear. The top row, containing the AFB amplicons, did not possess such clear and definite bands, despite earlier here and here having nice sharp bands. The roach trachea isolate never amplified with the AFB primers and we suspect it is a Bacillus spp.

The bottom row contained three of the transconjugates that grew after the pMarA tripartite conjugation. Only one of them had a nice band (maybe another in Lane 5?). This may confirm that what grew after the conjugation was indeed P. larvae, however, I'll need to still run a PCR to amplify the KanR transposon to determine if the transposon did indeed transfer to the P. larvae. 

Regardless, I am planning on re-running this PCR using the gDNA that I still have. Just as soon as I am able to locate more PCR super mix.

//EWW

Wax Worm/P. larvae LD50

From 3-7-15.

I set up another wax worm ingestion LD50 experiment again since I still had additional early instar worms. Using a newly created P. larvae 9545 spore stock that was only at a concentration of 1x10^5 CFU/mL I added 100 uL of several dilution in Bee Artificial Diet (BAD).

P. larvae spore dilutions were made in a five fold serial dilution (1:5, 1:25, and 1:125). There were 30 wax worm replicates for each dilution split into three different containers with 10 wax worms in each.


The wax worms in the containers were stored in the walk in 30C incubator inside of a Tupperware container. I will monitor the survival of these wax worms over the course of seven days.

/EWW

Detection of P. larvae in Local Honey

From 2-24-15.

After attempting different types of liquid media I determined that Nutrient Broth (NB) was most successful at propagating the growth of the six isolates: dd1-8a, dd5-Q, dd8-63a, dd11-2a,dd12-22a, and the roach trachea isolate. A single isolated colony of each isolate was taken from MYPGP agar and inoculated into 10 mL of NB in 50 mL conical tubes. Cultures were grown overnight at 37C shaking 225 rpm.

The Qiagen DNeasy Blood & Tissue kit was used to extract genomic DNA from each of the isolates. The gDNA is stored at -20C until use in PCR reactions.

//EWW

Wax Worm/P. larvae LD50

From 3-3-15.

Below is the survival curve of the wax worms fed different spore concentrations that were mixed with the wax pellets:

As discussed earlier, the wax worms exposed to the P. larvae spores mixed with the wax pellets didn't seem to result in disease after seven days. However, the 1:5 dilution of B. thur spores did. The rest of the data is stored in an Excel file.

It is Day 4 for the wax worms that were fed the spores in BAD that were not mixed with wax pellets. Already there are surprising differences between the previous experiment with the wax pellets and the one now without.

Wax Worms in the the B. thru 1:25 treatment group, notice the darker color running along the center of the wax worm.

Dead wax worm from the P. larvae 1:5 treatment group. Notice dried up. Perhaps this wax worm died earlier in the last 24 hours and may have had more time to dry?

Another dead wax worm from the P. larvae 1:5 treatment group. Not as dry as the one before, almost sticky looking and wet. Perhaps this is a more recent dead worm within the last 24 hours?

Wax worm from the P. larvae 1:5 treatment group. Has similar wounds and signs of disease that were seen in B. thur wax worms from the previous experiment from 3-3-15. 

//EWW

Wax Worm/P. larvae LD50

From 3-2-15.

Day 6
Checked the survival of the wax worms that were fed the bacterial spores. Once again, all the wax worms in the P. larvae spore treatment groups were accounted for alive and well. However, the wax worms in the B. thur. groups have begun to have visible signs of illness, as seen in the multiple images below. I would assume these blemishes on their bodies are due to ingestion of the B. thur spores. The wax worm bodies are covered in round dark wounds that almost look like burns.

"burns" on the outside of the wax worm

Dead wax worm (brown color)

Another wax worms with visible signs of disease

Below are several pictures of the wax worms from the P. larvae treatment groups for comparison. The wax worms were all fed spores at similar instars. The below wax worms do not possess the visible signs of disease.

Healthy wax worm from P. larvae treatment group

No visible signs of disease

Healthy wax worm

Since I still have left over BAD/spore dilution made up that have since been stored in the 4C fridge from 2-25-15 I set up the ingestion LD50 experiment again with a few modifications. Only the 1:5 and 1:25 dilutions were used from each of the spore stocks (P. larvae and B. thur) and no wax pellets were added to the containers. As before, 100 uL of the spore dilutions in BAD were added to the center of the small containers with 10 early instar wax worms to each treatment group. I want to see if there is a difference between adding the wax pellets and them not being present. I speculate that the presence of the wax pellets either dilute or nullify the effects of the spores to cause disease, or that only by having them there were the spore ingested at all (since the wax worms ate the wax that was coated in spores). Below is a picture of the set-up for this new experiment:

A cover was placed on each container and incubated in the 30C walk in incubator. I will continue to monitor the survival of each of these experiments.

/EWW

Wax Worm/P. larvae LD50

From 2-25-15.

Five days after wax worm exposure to spores. The only wax worms in the P. larvae spore groups that have died so far were on Day 1 and it was very apparent that those wax worms either drown in the added diet or were killed during the transfer to the smaller container (crushed bodies). There was wax worm that also died on Day 1 in the B. thur group as well and it was also very obviously due to handling errors during transfer. A table of the survival of each treatment group will be posted at the end of seven days.

Currently, the majority of the wax worms in the P. larvae groups appear to be in good health with white/cream colors, very active when prodded, and also have visibly grown in size after the last five days. It is evident that the wax worms have been eating the wax pellets since there are now much smaller wax pellets present in the containers. I assume this indicates that the wax had been ingested and processed by the wax worms. This is good, since the spores were mixed with the wax pellets initially. Interestingly, the majority of the wax worms have created some sort of make-shift cocoon using the wax pellets and the digested wax pellets. As evident by the images below. When the wax was removed from the tops of the wax worms they appeared to be in good health still with no visible signs of illness. All the wax pellets were initially in the center of the plate, they have since all made their way to the sides of the plate.

Healthy WW, can see digested wax pellets as well, the wax that was added was in the sphere shapes (also seen)

I have been observing increased deaths in the B. thur groups (1:5 and 1:25 dilutions). The wax worms in these treatment groups have not increased in size as much as the wax worms in the P. larvae treatment groups, nor are they as brightly colored (they are more grey/black) or as lively. One of the wax worms in the 1:5 group turned completely black and has begun to liquify.

It should be noted that there was a higher spore concentration in the B. thur groups that was fed to the wax worms than in the P. larvae groups:

The 1:5 B. thur group = 2x10^7 spores

The 1:5 P. larvae group = 2x10^3 spores

I will continue to monitor the survival of the wax worms in each of the groups as well as investigate ways to increase the spore yield from P. larvae. The increased death in the B. thur groups compared to the P. larvae groups is likely related to the amounts of spores that were being fed to the wax worms. I simply do not have a high enough P. larvae spore concentration to caused death in the wax worms at this time.

//EWW

Wax Worm/P. larvae LD50

Goal: Create and LD50 curve of P. larvae spores using early instar wax worm larvae. This information will be important in determining if wax worms can be used as surrogates to the honey bee larvae. This information will also be used in a future grant proposal. 

The P. larvae spore stock G (1-31-15) that has since been stored at 4C in ddH2O was serially diluted 1:5 in Bee Artificial Diet (BAD) made last summer. The spores were diluted down to 1:3125. 

To set up the inoculation, the wax worms will be attempted to be fed the spores through the BAD. For this, small plastic containers (seen above) with covers will be used. About 150 mg of wax pellets were added to the center of each container using a metal scoopula. A volume of 100 uL of the diluted spores in BAD was added to the top of the wax pellets and mixed together using a sterile toothpick (picture seen below).

Mixing in the BAD/spores with a tooth pick

A total of 10 wax worms were added to each container. There were duplicate containers made of each spore dilution (2 biological replicates with 10 technical replicates each). Negative controls included a "no diet", where the wax worms were placed only on the wax pellets, and a "no bacteria", where the wax worms were introduced only to the BAD with no spores. Bacillus thuringiensis spores (a known insect pathogen) was used as a positive control (diluted 1:5 and 1:25).

10 WW to a single container

The containers were placed inside a tupperware container and placed in the walk in 30C incubator (where the wax worms were originally taken from). I will check the survival of each of the containers over the course of a week and hopefully be able to put together an spore ingestion LD50 if this is indeed a viable method of determining.


//EWW